Nonmuscle myosin 2 (NM2) molecules carry out a wide variety of functions within cells. There are three NM2 heavy chain genes. We are expressing full length NM2 in the baculovirus Sf9 system. We are studying their filament structure and how phosphorylation of both the heavy chain and light chain affects filament formation. We use a single filament motility assay system wherein we can image the movement of fluorescently labeled myosin filaments over actin filaments fixed to the surface. We are examining the copolymerization of NM2A and NM2B form co-polymers in vitro. We are collaborating with the Korn lab at NHLBI and the lab of Laci Nytray at Eotvos Lorand University in Budapest to study the effects of heavy chain phosphorylation on filament assembly. Optical trapping studies reveal that NM2A and NM2B are not processive as single molecules. Bipolar filaments of NM2B containing about 30 myosin molecules move processively along actin filaments attached to the surface. By copolymerizing full length NM2B with a Halo-capped tail fragment of NM2B, we show that between 5 and 8 motor domains per half filament are required for processive movement. Surprisingly, filaments of NM2A do not move processively under these same conditions which may be due to the lower duty ratio of this myosin compared to that of NM2B. NM2A can be co-polymerized with NM2B molecules and these heterotypic filaments move processively provided sufficient NM2B is present. In the presence of 0.5% methylcellulose which mimics the viscosity of the cytoplasm both NM2A and NM2B filaments move processively. We can also image the movement of unlabeled NM2 filaments using iSCAT microscopy. We have reexamined the activity of an N93K mutant of NM2A which we previously published was inactive with regards to ATPase activity and in vitro motility. Recent work suggests that if properly expressed in Sf9 cells, the protein has substantial ATPase activity. We believe that the reason for the previous determination of inactivity may be related to problems in properly folding the molecule and this may explain some of the disease phenotypes in humans bearing this mutation. In collaboration with others, we have determined the 2.25 Angstrom structure of a motor domain of NM2C and have shown that there is an allosteric communications pathway that operates from the distal end of the motor domain to the active site via a positively charged residue present at the interface between the N-terminal subdomain, the converter and the lever arm. In collaboration with the Nyitray lab we showed that GFP-capped myosin tail fragments formed bipolar filaments similar to those formed by full length myosin. S100a is a small calcium binding protein that is upregulated in metastatic cells. This protein has previously been shown to bind to the distal tail of NM2A with high affinity. Its affinity for NM2B is 3 orders of magnitude less. S100a addition to preformed NM2A/NM2B co-filaments results in the extraction of NM2A from these filaments. Thus in a cell, NM2A could effectively sequester NM2A in the presence of calcium while leaving NM2B in filamentous form. We have recently crystallized the motor domain and the light chain binding region of NM2B and are solving its structure. We collaborated with the laboratory of Philipp Kukura who developed a novel interferrometric scattering mass spectrometry (iSCAMS) microscope which determines the molecular mass of protein or protein complexes which land on a microscope surface. The light scattering from proteins on the surface was shown to be linearly proportional to their mass from 40 kDA to 800 kDa. We showed that protein shape does not affect the scattering intensity since NM2 that is in the folded, off state with a molecular length of 60 nm scatters the same amount of light as does the same myosin in its filly extended conformation (150 nm). This instrument will be used to determine the equilibrium concentration of associating proteins.